CANCER:

Probing cellular metabolism enables scientists to elucidate how enzyme molecules work together to keep the cell running. It also enables scientists to test the efficacy of anti-cancer drugs.

Such experiments are challenging. They just got easier, thanks to Itamar Willner and coworkers at The Hebrew University of Jerusalem. These scientists have found a way to turn nanoparticle fluorescence on and off in response to metabolic activity.

Principles of metabolic probing.

The scientists' nanoparticles are comprised of a cadmium selenide/zinc sulfide core (quantum dots), coated with a "spacer" layer of inert protein molecules. Nile blue molecules are affixed to the periphery.

Under normal conditions, these nanoparticles are not fluorescent. Although the quantum dots emit fluorescence, the Nile blue molecules absorb it, without emitting the energy as fluorescence.

The nanoparticles are designed such that they are fluorescent when metabolism is proceeding. How can the fluorescence be turned on?

1. Many metabolic processes utilize a molecule abbreviated as NAD⁺, 1,4-nicotinamide adenine dinucleotide. In many processes, NAD⁺ is converted to NADH.

2. NADH can provide Nile blue molecules with an electron. When this happens, the Nile blue molecules are no longer able to absorb fluorescence in the visible region.

3. Consequently, the quantum dot core of the nanoparticles can then emit fluorescence. Thus, in the presence of NADH molecules, the nanoparticles are fluorescent.

4. Therefore, if fluorescence is observed, one knows that an NAD⁺-dependent process is going on. By inference, metabolism is proceeding.

5. Furthermore, the intensity of the fluorescence tells you the concentration of NAD⁺ molecules present, This enables one to quantitate the level of activity of the enzyme involved.

Visualizing metabolism with nanoparticles.

The scientists first set out to determine the activity of the enzyme alcohol dehydrogenase, in a test tube, with their nanoparticles. Alcohol dehydrogenase converts ethanol molecules to acetaldehyde molecules, and in so doing converts NAD⁺ molecules to NADH molecules.

As the scientists increased the concentration of ethanol, the enzyme went to work, resulting in an increased production of NADH molecules. Consequently, due to an inactivation of the Nile blue molecules affixed to the nanoparticles, the fluorescence intensity of the nanoparticles increased.

This demonstrates that the scientists' nanoparticles can detect and quantify enzymatic function. They then set out to test their nanoparticles in cancer cells.

Visualizing cancer cell metabolism with nanoparticles.

Cancer cells exhibit an enhanced level of metabolic activity. They are therefore prime targets for the scientists' nanoparticles to probe their behavior.

After D-glucose was added to a suspension of HeLa cells (an immortal cervical cancer cell line) that had previously taken up the scientists' nanoparticles, the cells began metabolizing the glucose. This metabolism led to an increased production of NADH molecules, which caused the scientists' nanoparticles to emit fluorescence.

When L-glucose was added to the cell suspension, the cells could not metabolize it. This lack of metabolism, and the consequent lack of NADH molecules produced, prevented fluorescence emission from the nanoparticles.

The cells were fluorescent when they metabolized D-glucose, but were not fluorescent when they could not metabolize L-glucose. Therefore, the nanoparticles are an effective reporter of cellular metabolism.

Visualizing cancer drug efficacy with nanoparticles.

Can the same principles be applied towards monitoring the efficacy of anti-cancer drugs? This is what the scientists set out to investigate next.

Cancer cells exhibit reduced metabolic activity when they are being killed by drugs. This cell death can be monitored by the scientists' nanoparticles.

After taxol, an anti-cancer drug, and D-glucose were added to a suspension of A549 cells (skin cancer cells) that had previously taken up the scientists' nanoparticles, the cells began to die. This lack of metabolism, and the consequent lack of NADH molecules produced, prevented fluorescence emission from the nanoparticles.

The cells were less fluorescent when they were being killed by anti-cancer drugs. Therefore, the nanoparticles are an effective reporter of anti-cancer drug efficacy.

Limitations of the nanoparticles.

The scientists note that they cannot reproducibly control the fluorescence properties of the nanoparticle core from batch to batch, and cannot precisely control the amount of Nile blue molecules affixed to the nanoparticles. This means that fluorescence intensities observed from one batch of nanoparticles cannot be directly compared to those from another batch of nanoparticles.

However, when these problems are solved, the scientists will have a rugged and versatile tool to probe cellular metabolism and the efficacy of anti-cancer drugs in living cells. Such tools are sorely needed by many scientists, especially those who need to quickly probe cellular function in clinically relevant model systems.

for more information:
Freeman, R.; Gill, R.; Shweky, I.; Kotler, M.; Banin, U.; Willner, I. Biosensing and probing of intracellular metabolic pathways by NADH-sensitive quantum dots. Angew. Chem. Int. Ed. 2009, 48, 309-313.